October 19, 2015

This post is part of a weekly series about image screening. All published posts can be found on this page.

One component of the production process at many journals is to screen all images to confirm that they are of sufficient resolution for publication and to ensure that they have not been altered in any way that could impact the paper’s conclusions. We’ve discussed the importance of figure and image resolution. Part of ensuring the appropriate resolution when acquiring images lies in checking the settings on your imager and/or scanner. The second, equally important part comes when you are ready to export a gel image to a non-proprietary file type. Which one to choose? JPEG? TIFF? BMP? PNG?

JPEG or JPG (short for Joint Photographic Experts Group) is a compression technique that reduces file size and is therefore widely used for large image files. However, it is “lossy”, which means that some amount of data is lost in the compression process. In contrast, TIFF/TIF (tagged image file format) is a lossless format that, unlike JPEG, allows you to edit and re-save the file without any loss in image quality. We therefore wholeheartedly encourage you to use TIFF only. Both JPEG and TIFF are widely supported formats, so you should be able to obtain these files from scanners and digital imagers and to open them with your figure-making software. If for any reason your data has to be acquired in a JPEG or any other non-TIFF format such as BMP, one way to avoid future loss of resolution is to open it immediately (in Photoshop for instance) and save or export the image as a TIFF.

Now that you have high-resolution image data properly stored as TIFFs, it is time to start making figures (and to write the actual paper! And do more experiments! And draft this grant/fellowship application!)

October 12, 2015

#3. If you’re reading this, it’s not too late: how to get image data of appropriate quality and resolution

This post is part of a weekly series about image screening. All published posts can be found on this page.

The key to high-quality figures is high-quality data capture. Figure-making settings are important, but so are the settings used during digital capture and storage of images. As discussed previously, it sometimes happens that pieces of image data in a figure are not of sufficient quality to allow our routine figure-screening process. Researchers should therefore carefully consider the resolution settings when first acquiring and storing their data. Western blot and gel images in particular are a contentious subject, but they do not have to be! Here are some easy ways to resolve (and hopefully prevent) any issue you might encounter during a journal’s figure check.

Whether you are using films or a digital imager, there are two crucial steps to pay close attention to: scanning and exporting. It is very important to do both at the highest resolution available to you (minimally 300 dpi) and to make sure that no pixel averaging is enabled by default (more on that in the next paragraph). This likely means you will have to explore and adjust the system’s settings at the time of first use so as to ensure scanning and exporting generates images at 300 dpi. If the resolution setting is not easily identifiable or seems as though 72 dpi is your only choice, we recommend calling a service representative, your IT department (or even us and we’ll try our best to help).

In addition to choosing the appropriate resolution, you must check that no averaging is enabled during scanning or exporting. “Pixel averaging” is a common way of reducing noise and file size in image processing. What this process does is apply an algorithm that “averages” a group of pixels to produce “larger” new pixels and will result in fewer pixels overall. In other words, each new pixel is a function of an area of pixels. This process implies that you are losing some of the information that the original, “smaller” pixels present in the image encoded. That is why, although you may want to play with it for your next Instagram art project, pixel averaging should be avoided for all image data.

Lastly, when exporting, it is important to export to a loss-less file type such as TIFF, and avoid JPEGs at all cost – more details to come in next week’s installment.

Image size can be defined as the total number of pixels in an image and is therefore routinely given in pixel dimensions: pixel width x pixel height. Size can also be given in inches, mm, cm, picas, etc.

Image resolution refers to the level of detail of an image, or in other words, pixel density. For digital files, it is commonly measured in dpi (dots per inch: the number of dots per inch of a printed image) or ppi (pixels per inch).

As indicated by these units, resolution depends on size. Opening an image in Photoshop or any Adobe software will allow you to discover what its size and resolution are by clicking on “Image size” in the “Image” menu; the information will be displayed in various units.

the size of the figure should be comparable to that of the published, printed figure (about 7 inches high x 9 inches wide);

the figure file itself should be at a resolution of 600 dpi (this is higher than the images that go in it so that their resolution is preserved and it is necessary to meet printing requirements);

each individual image copied or imported into the figure file should also originally be at a resolution of 300 dpi at least (more on how to obtain 300-dpi image data in our next post).

For example, here is a quick video showing how to set up a figure file using Adobe Illustrator,

Beware: your program’s default setting for a new document may be only 72 dpi! When creating a new figure file in Photoshop, Illustrator, or any other software, you must first check its resolution in a program such as Photoshop and set it at 600 dpi. If you’re using a “vector” program, such as Illustrator, you can instead set the Profile to “Print quality” or rasterizing effects to “High.” Can’t find these settings in PowerPoint? This is one of the main reasons we discourage its use -- it is not designed to preserve print quality and high resolution, and we’ll explore the differences in software in a future post in this series.

Setting the resolution initially is very important as a lower resolution figure file means losing some of the quality of the pieces of image data you are placing into the file and displaying in the figure. It is essential to pay attention to resolution from the start as, unfortunately, “resampling” in Photoshop does not yield true high-resolution images -- in other words, manually changing the resolution of a 72-dpi file to 300 dpi will not make it of acceptable resolution and quality, and the figure will most likely fail a routine production screening.

How can you deal with images that have been acquired or stored at less than 300 dpi? Come back next week to find out!

You may have heard: JCB has high image standards. Part of JCB’s and many other journals’ routine production process involves screening all editorially accepted manuscripts to confirm that images are of sufficiently high resolution for publication and to ensure that they have not been over-adjusted or manipulated in any way that could impact the conclusions of the work. Sometimes, figures fail this screening, or, more often, are not of sufficient quality to allow this screening process. But what are these researchers doing wrong? Not that much, actually. It turns out that, in the vast majority of cases, a few simple fixes can make a figure JCB-ready. It doesn’t take much time or effort, but it does require that you know a few golden rules about quality image preparation. As scientific editors at Rockefeller University Press journals, we work every day with our production editors to move manuscripts through this screening, and in this series, we’ll share with you what you need to know about image data acquisition and storage, as well as figure preparation. We hope it will be useful if you wish to submit your work to a journal that screens figures or if you just want to learn more about the process and earn bragging rights.

September 14, 2015

In the latest issue of JCB, Wynne and Funabiki reveal that, in the absence of microtubule attachments, a subset of kinetochore proteins form expanded structures that may help activate the spindle checkpoint and capture microtubules. As described in this week’s In Focus, treating Xenopus egg extracts with the microtubule-depolymerizing drug nocodazole causes multiple kinetochore proteins – including the spindle checkpoint regulators BubR1 and Mad1, as well as proteins, such as CENP-E and dynein, involved in lateral microtubule attachment – to form long, thin filaments that extend more than a micron away from the centromeres of mitotic chromosomes.

Kimura et al. describe how TRIM family proteins target specific inflammatory regulators for degradation via the autophagy pathway. As described here, TRIM20 targets several subunits of the inflammasome for degradation by linking them to key autophagy regulators such as ULK1 and Beclin1. TRIM21, on the other hand, links the transcription factor IRF3 – an activator of type I IFN responses – to the autophagy machinery.

Von Büdingen et al. reveal that a component of the myelin sheath surrounding spinal cord axons regulates the growth of neighboring unmyelinated neurons by sequestering NGF. NGF binds with high affinity to a myelin component called MOG, limiting its ability to stimulate the growth of unmyelinated pain-sensing neurons. MOG-deficient mice show increased sprouting of pain-sensing neurons, an observation that could help explain why patients suffering from the demyelinating disease multiple sclerosis often experience chronic neuropathic pain. More here.

Zhou et al. reveal that the disease-related protein progranulin gets a “piggyback” ride to lysosomes by binding to another lysosomal protein, prosaposin. Progranulin, which is mutated in both frontotemporal lobar degeneration and neuronal ceroid lipofuscinosis, can be transported to lysosomes by the sorting receptor sortillin. But, as summarized here, Zhou et al. describe an alternative route, in which prosaposin links progranulin to another sorting receptor, namely the cation-independent mannose 6-phosphate receptor.

Meanwhile, Paul et al. describe how actin spikes drive 3D cell migration, and Lang et al. explain how defects in ER-mitochondrial contacts can be bypassed by other organelle contact sites. We interview the senior authors of both papers in this month’s biobytes podcast; listen below or subscribe in iTunes!

September 08, 2015

On Tuesday morning, at the 2015 EMBO meeting in Birmingham, UK, I attended the session on the ER and protein folding. The session – organized by Manu Hegde – illuminated a lot of the molecular details of how proteins can be translocated into, or extracted from, the endoplasmic reticulum. Irmgard Sinning described the crystal structures of some of the RNA and proteins that make up the signal recognition particle, helping to explain how this complex can arrest the translation of nascent secretory proteins and deliver them, along with the ribosome to the ER membrane. Hegde, meanwhile, talked about how, once docked at the ER membrane, the ribosome-nascent chain complex might open up the translocon pore to allow the secretory protein’s passage into the ER.

The session followed on from Monday’s keynote lecture by Peter Walter, who presented a wonderful example of how basic, curiosity-driven research can quickly lead to unexpected clinical applications.

Walter and colleagues initially screened for small molecule inhibitors to block the PERK pathway, a component of the Unfolded Protein Response to ER stress that reduces general protein translation while upregulating the translation of a transcription factor, ATF4, that can help cells either adapt to this stress or undergo apoptosis. The researchers identified a molecule called ISRIB, which blocked ATF4 induction and restored general translation by boosting the activity of the translation initiation factor eIF2a. The activity of this initiation factor is implicated in memory consolidation and, remarkably, ISRIB enhances learning and memory in mice by inhibiting long-term depression. ISRIB has quite favorable pharmacological properties, and Walter thinks that it might be able to treat a variety of cognitive disorders in humans. A serendipitous discovery, for sure, but one that highlights the importance of basic cell biological research.

September 07, 2015

The sixth annual EMBO meeting is well underway here in Birmingham, UK. There have been a lot of great talks so far, and I hope I’ll be able to blog about some of them over the next few days.

One of the main subjects at this meeting has been unconventional RNAs i.e. RNAs that don’t fall into the classical categories of mRNAs, tRNAs, or rRNAs, such as micro RNAs or long, non-coding RNAs generated from retrotransposons and other repeat elements in the genome. As many of the speakers pointed out, “unconventional” is becoming something of a misnomer, as researchers appreciate how much of the non-coding genome is transcribed to produce functionally important RNAs. Retrotransposons, for example, make up 45% of the human genome.

On Saturday evening, Joan Steitz gave the meeting a wonderful opening lecture, presenting two recently published stories. She described how osmotic stress activates a signaling pathway that induces the transcription of a whole new class of long, non-coding RNAs from DNA sequences Downstream of Genes (hence the name DoGs). These RNAs, often more than 45 kb in length, seem to bind back to these chromatin regions, and may help to maintain the integrity of the nucleus during osmotic stress. Steitz also described the function of a non-coding RNA produced by the Epstein-Barr virus. This RNA, EBER2, works in an unusual (or unconventional!) way. It binds to nascent RNAs transcribed from the terminal repeat regions of the integrated viral genome, and then recruits a host cell transcription factor, PAX5, so that it can silence nearby latency-promoting genes, and promote lytic viral replication.

On Sunday, an entire session was dedicated to the subject of unconventional RNAs. Javier Cáceres talked about the post-transcriptional regulation of miRNA biogenesis, in particular how variations in the sequence of a miRNA can affect its expression level. Jernej Ule described how RNA-binding proteins can regulate the expression and function of transcripts produced from retrotransposons, while Robert Martienssen discussed how small RNAs contribute to transposon silencing and epigenetic inheritance in Arabidopsis. And Grzegorz Kudla described a nifty method to map physical RNA-RNA interactions across the transcriptome, as well as a way to look at epistatic interactions both within and between RNA genes.

It really is a fascinating field. Unconventional RNAs can do so many different things and I’m sure we’re only seeing the tip of the iceberg. It looks like RNA biology will continue to break conventions for some time to come.

August 31, 2015

In today’s new edition of JCB, Carroll-Portillo et al. reveal that mast cells form physical contacts with dendritic cells to facilitate the transfer of antigens inside exosomes. The dendritic cells can then present these antigens to T cells in order to stimulate an immune response. You can learn more about this new type of immune cell interaction in this week’s In Focus.

Hong et al. demonstrate that the phospholipid PI(3,5)P2 regulates the dynamics of branched actin networks on the surface of endosomes. As explained here, the phospholipid binds to the actin regulatory protein cortactin, removing it from branched actin filaments so that their stability is decreased. In the absence of PI(3,5)P2, therefore, cortactin accumulates on late endosomes and actin turnover is reduced.

DeGeer et al. describe how the chaperone protein Hsc70 shepherds a key guanine nucleotide exchange factor to the tips of growing axons. Hsc70 delivers the exchange factor Trio, which activates the small GTPase Rac in response to the axonal guidance cue netrin-1. Embryonic mice expressing a defective form of Hsc70 fail to undergo normal brain development. More here.

Xia et al. describe a way to force cancer cells to destroy a key metabolic enzyme they need to survive. As described here, the researchers find that simultaneously treating cancer cells – but not healthy cells – with inhibitors of both autophagy and the receptor tyrosine kinase FLT3 induces the degradation of hexokinase II, which catalyzes the first step of glycolysis.

Meanwhile, Schweizer et al. describe how a membranous spindle envelope helps numerous proteins accumulate around the mitotic spindle in order to facilitate bipolar spindle assembly and accurate chromosome segregation. Paul Maddox and Anne-Marie Ladouceur comment on the paper here, while senior author Helder Maiato discusses his group’s findings in this month’s biosights video podcast, which you can watch below or subscribe to in iTunes.

And finally, as always, don’t forget to check the JCB table of contents to see all the other papers published in today’s issue!

August 17, 2015

Time for a quick roundup of some of the highlights from the latest issue of JCB…

Lawrimore et al. reveal that loops of pericentric chromatin repel each other to generate tension between sister centromeres independently of the mitotic spindle. As explained in this week’s In Focus, the structure of this pericentric chromatin spring may also allow it to act as a shock absorber that can buffer the variable forces generated by dynamic spindle microtubules.

Henne et al. reveal that a protein linked to human neurological disease helps tether vacuoles to the ER in budding yeast. Mutations in human sorting nexin 14 cause an autosomal-recessive form of cerebellar ataxia. Henne et al. find that similar mutations in the yeast homolog, Mdm1, disrupt this protein’s localization to ER-vacuole contact sites, which appears to result in impaired sphingolipid metabolism.

Zhao et al. reveal that the microRNA miR-7 suppresses gastric cancer by inhibiting pro-oncogenic NF-kB signaling. The researchers also find, however, that the NF-kB pathway can inhibit miR-7 expression as part of a feedback signaling loop. Chronic Helicobacter pylori infection is a major risk factor for gastric cancer, and co-culturing the bacterium with gastric epithelial cells activated NF-kB signaling and downregulated miR-7, a potentially key step in gastric cell transformation. More here.

And Hendrix et al. use a series of fluorescence fluctuation imaging techniques to reveal that HIV-1 particles start assembling in the cytoplasm of infected cells. As summarized here, the viral polyprotein Gag starts to oligomerize on viral RNAs in the cytoplasm, before the particles associate with the plasma membrane to complete their assembly into new viral particles.

Meanwhile, in this month’s biobytes podcast, you can hear author Vibe Oestergaard describe how a protein called TopBP1 protects the genome during mitosis (Pedersen et al.), and David Williams explain how defects in phagosome transport lead to age-related macular degeneration (Jiang et al.). Listen below or subscribe in iTunes!

July 20, 2015

In the latest issue of JCB, Strale et al. reveal that interactions between neighboring E-cadherin molecules help to strengthen their connection to the actin cytoskeleton and stabilize cell-cell contacts. As explained here, E-cadherin mutants unable to interact with other E-cadherin molecules in the same membrane can still form intercellular adherens junctions, but these junctions are weaker and cannot coordinate the movements of cells undergoing collective cell migration.

Donovan and Bretscher track the behavior of individual secretory vesicles in budding yeast in order to provide a timeline of the events involved in exocytosis. As detailed in this week’s In Focus, the researchers find that vesicles consistently tether to the bud cortex for 18 seconds before they fuse with the plasma membrane. This tethering period, which may allow cells to ensure vesicles are targeted to the right location, is regulated by the Rab GTPase Sec4p and the myosin motor Myo2p. The researchers also find that Myo2p dissociates from the vesicles about 4 seconds before fusion, in contrast to other components, such as the exocyst complex, that remain bound to vesicles until exocytosis is completed.

Nyathi and Pool describe how a chaperone complex called NAC helps ensure nascent polypeptides are correctly processed as they emerge from ribosomes. Many of the factors that modify, fold, or target nascent polypeptides bind to a particular region of ribosomes called the Universal Adaptor Site. As summarized here, Nyathi and Pool find that NAC regulates the competition between two processing factors: Map1, an enzyme that cleaves the N-terminal methionine off of most cytosolic proteins, and the SRP, which guides nascent secretory and membrane proteins to the ER. Moreover, NAC also prevents nascent secretory proteins from aggregating before they can be recognized by the SRP.

Yan et al. describe how the deubiquitinating enzyme complex BRISC regulates the mitotic spindle assembly factor NuMA. In the absence of BRISC, mitotic cells form disorganized, multipolar spindles because ubiquitinated NuMA shows an increased association with two of its regulators, importin-beta and dynein. More here.

Elsewhere, Bamidele et al. describe how cells regulate the endocytic trafficking and function of the CXCR4 chemokine receptor, and Treuner-Lange et al. explain how a small GTPase combines with bacterial actin to regulate bacterial focal adhesions and motility. You can hear the senior authors of both papers discuss their findings in this month’s biobytes podcast. Listen below or subscribe in iTunes!

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